A so-called endotoxin (intracellular toxin) is a major component of cell membranes of bacteria that are not stained by Gram staining (Gram-negative), and contains a lipid-polysaccharide called a lipopolysaccharide, specifically a lipopolysaccharide (LPS) in which a lipid called Lipid A and a polysaccharide chain are bound via 2-keto-3-deoxyoctonate (KDO). A lipid structural component called Lipid A included in the lipopolysaccharide, when entering a human body by infection, binds to a cellular receptor, causing inflammation, and causing a variety of severe clinical symptoms in many cases. As described above, the endotoxin is a substance causing clinical symptoms such as sepsis and bacteremia that are very high in fatality. Thus, estimation of the endotoxin which has entered the body is highly demanded clinically.
In addition, it is important that a medicinal product (such as an injection), a blood preparation, a medical device (such as an angiocatheter), a large amount of purified water, or the like is not contaminated with the endotoxin (pyrogen-free). Moreover, it is essential to properly remove or control the endotoxin in a medicinal product (such as a recombinant protein or DNA used for a gene therapy), a food addictive, a cosmetic, or the like prepared by using bacteria.
In confirmation of the removal of the endotoxin or measurement of the endotoxin in emergency medicine, promptness is required for attaining the purposes of coping with a large number of measuring samples and carrying out life-saving treatment.
Research has been made since old days on measuring an endotoxin value for treating sepsis or the like. Since the discovery of the phenomenon that a group of factors contained in a limulus amebocyte lysate each specifically react with the endotoxin, resulting in an aggregate which covers a wound, trials for quantifying the endotoxin have been made by using the limulus amebocyte lysate (LAL reagent or limulus reagent).
A first measurement method involving using the limulus reagent was a semiquantitative measurement method called a so-called gelation method, in which plasma from a patient serving as a sample is mixed with the limulus reagent, the mixture is left to stand still, the mixture is positioned upside down after a certain time, the presence or absence of gelation is confirmed by whether or not the solution is solidified, and the amount of an endotoxin is estimated based on the maximum dilution ratio at which the gelation is caused.
After that, attention has been paid to an increase in turbidity in the course of a gelation reaction. As a result, there is known a turbidimetric time assay, in which a turbidimeter using an optical measurement method is used to quantitatively measure an endotoxin concentration based on a change in turbidity involved in a gelation reaction of a mixing solution left to stand still.
In addition, a synthetic chromogenic substrate method has already been known, in which a gelation reaction causing conversion from coagulogen to coagulin is replaced by a chromogenic reaction of a synthetic substrate in the final stage of a reaction process with a limulus reagent. This method is a method in which a synthetic chromogenic substrate (Boc-Leu-Gly-Arg-p-nitroanilide) is added in place of a coagulation precursor (coagulogen) in a coagulation process, hydrolysis of the synthetic chromogenic substrate then produces free p-nitroaniline, and colorimetric analysis of the resultant yellow chromogenic development is performed to measure a coagulogen decomposition ability, thereby measuring an endotoxin concentration.
Further, as conventional endotoxin measurement methods, for example, ones described in Patent Literatures 1 to 5 have already been employed.
Patent Literature 1 is a method of stabilizing an endotoxin in an aqueous solution, the method including causing a water-soluble protein that has an affinity for the endotoxin and does not have such property as to inhibit or accelerate a reaction between the endotoxin and a horseshoe crab amebocyte lysate to coexist in the aqueous solution containing the endotoxin to accurately measure an endotoxin concentration in the aqueous solution over along time period.
Patent Literature 2 is a method including causing a peptide derivative (or protein) having such property as to bind to an endotoxin to suppress the activity of the endotoxin and a surfactant to coexist in a sample containing the endotoxin to suppress the activity of the endotoxin.
Patent Literature 3 is a method including mixing a horseshoe crab amebocyte lysate and a sample containing an endotoxin or a β-D-glucan in the coexistence of a predetermined water-soluble polymer, measuring a time period required for the degree of an optical change to reach a predetermined value, and determining the amount of the endotoxin or the like in the sample on the basis of a correlation between the obtained time period and the amount of the endotoxin or the like to detect the endotoxin or the like by means of a turbidimetric time assay with high sensitivity.
Patent Literature 4 is a measurement apparatus for measuring a target substance in a sample through a gelation reaction to accurately measure the concentration of a substance to be measured by the gelation reaction such as an endotoxin or a β-D-glucan within a short time period, the apparatus being a technology in which a sample cuvet storing a solution containing an analyte containing the substance to be measured and a reagent causing gelation is irradiated with illuminating light from a light-emitting diode, the solution in the sample cuvet is agitated with a stirrer bar to produce minute and uniform gel particles, and is then passed through the illuminating light, transmitted light from the gel particles produced in the sample cuvet is detected with a diode, and the concentration of the substance in the solution is measured on the basis of the transmitted light detection output.
Patent Literature 5 is a technology including a sample cuvet, a reagent that is stored in the sample cuvet in advance and reacts with a target substance in a sample to gelate, a stirring member that is stored in the sample cuvet in advance and stirs a mixing solution formed of the injected sample and reagent to suppress the gelation of the entirety of the agitated solution, and a sealing member which seals the opening of the sample cuvet in a state where the reagent and the stirring member are stored in the sample cuvet, and through which the sample can be injected into the sample cuvet after the sealing, in which a stirring operation by the stirring member is started at the time point at which the sample is injected into the sample cuvet and gel particles are produced in a state where the gelation of the entirety of the agitated solution is suppressed.
Further, the measurement technology using a gelation reaction is employed for measuring not only the endotoxin described above but also a β-D-glucan or the like.
The β-D-glucan is a polysaccharide constituting cell membranes specific to fungi. Measurement of the β-D-glucan is effective for screening a wide variety of fungi responsible for fungal infection, including not only fungi found in a general clinical environment, such as Candida, Aspergillus, or Cryptococcus, but also fungi rarely found in the general clinical environment.
The phenomenon that the gelation of the horseshoe crab amebocyte lysate is caused by the β-D-glucan is employed in the measurement of the β-D-glucan as well. The measurement is carried out by the above-mentioned gelation method, turbidimetric time assay, or synthetic chromogenic substrate method.
Measurement techniques for the endotoxin and the β-D-glucan have common points. For example, almost the same kind of measurement hardware is used to remove a Factor G component, which specifically reacts with the β-D-glucan, from the horseshoe crab amebocyte lysate, and hence a gelation reaction or chromogenic reaction selective for the endotoxin can be measured. Alternatively, the endotoxin in a sample is inactivated by pretreatment, and hence a gelation reaction or chromogenic reaction selective for the β-D-glucan can be measured.    [PTL 1] JP 2817606 B2 (Configuration of Invention)    [PTL 2] JP 3106839 B2 (Configuration of Invention)    [PTL 3] JP 3327076 B2 (Configuration of Invention)    [PTL 4] WO 2008-139544 A1 (Embodiment of Invention and FIG. 1)    [PTL 5] JP 2010-085276 A (Best Mode for carrying out Invention and FIG. 1)